Biology subjects

Bull, R. A.

Publications and source records attributed to Bull, R. A..

10 recordsLinked to original sources

bioRxiv2026

DENcode: A model for haplotype-informed transmission probability of dengue virus

Dengue virus transmission networks are often only partially resolved, due to gaps in sampling, unobserved mosquito-mediated transmission, and using methods (phylogenetics) that describe evolutionary relatedness but not explicit, probabilistic transmission links between individual infections. We developed DENcode, a framework to estimate the relative likelihood of vector-mediated transmission between pairs of dengue cases by combining a temperature- and time-modulated epidemiological kernel, which captures the extrinsic incubation period and human infectiousness, with a phylogenetically informed genetic similarity kernel derived from patristic distances between viral haplotypes or consensus sequences. Validation with a real-life dataset of 90 dengue infections sampled from Colombo, Sri Lanka between 2017 - 2020 and sequenced to resolve within-host haplotypes, DENcode estimates were stable across 100 Monte Carlo iterations, yielding narrow credible intervals (median width <0.001) and consistent top-ranked transmission pairs. Sensitivity analyses using ablation experiments showed that removing either the genetic or epidemiological component substantially altered the distribution of linkage probabilities, indicating that both contribute meaningfully to the inferred transmission structure. Serotype-specific transmission networks constructed from pairwise linkage probabilities from DENcode were analysed using degree- and path-based centrality measures at probability thresholds of 0.1 and 0.5, revealing relative importance of cases to disease transmission within the community. Haplotype-derived networks were more informative than consensus-based networks (x 3.6 and x 1.6 times more edges for DENV2 and 3 respectively). DENcode is a robust framework to explore dengue transmission within a community that provides an output of network of transmission probabilities informed by pathogen genetic similarity and clinical epidemiological parameters. Author summaryTracing epidemics of dengue in setting where dengue transmission happens continuously poses many challenges especially with limited availability of genomic surveillance. Here we introduce a model that uses genomic data together with time and location data to calculate a probability of two cases of dengue being related to each other. Using data from the Colombo dengue study, from 2017 to 2020, Sri Lanka, we evaluated the model. We used haplotype level sequences that correspond to the viral variation within the human host and consensus level sequences that average the data from a single human host into a single sequence. We constructed transmission probability networks for each dengue serotype and were able to identify patients who played key roles in the corresponding networks. We were able to show that this model is robust and will be a valuable tool in the context of dengue control.

bioinformatics↗

bioRxiv2024

Temporal dynamics of viral fitness and the adaptive immune response in HCV infection

Numerous studies have shown that viral variants that elude the host immune response may incur a fitness expense, diminishing the survival of the viral strain within the host, and the capacity of the variant to survive future transmission events. However, this generic definition can be further divided into two categories where intrinsic fitness describes the viral fitness without the influence of any immune pressure and effective fitness considers both intrinsic fitness with the influence of host immune pressure. Furthermore, co-occurring mutations outside the epitope regions targeted by the immune response may increase or decrease the likelihood of survival of the variant (known as epistasis). Analysis of viral fitness and epistasis over the non-structural protein regions is lacking for hepatitis C virus (HCV). Here, using a rare cohort of subjects very recently infected with HCV, we build upon our prior investigations by integrating mathematical modelling and experimental data to examine the interplay between the evolving transmitted/founder (T/F) viruses, the adaptive immune response, viral fitness, and co-occurring mutations. We show that viral fitness decreases during the first 90 days post-infection (DPI) associated with the magnitude of CD8+ T-cell responses and the initial level of diversification. Thereafter, viral fitness rebounds in a complex pattern of evolution characterized by multiple sets of co-occurring mutations. Finally, we show that an early and strong CD8+ T-cell response in the absence of neutralizing antibodies (nAbs) imposes a strong selective force on the T/F virus population, enabling the virus to escape and establish chronic infection. Understanding these dynamics is highly relevant for HCV vaccine design and supports a vaccine strategy that induces broad immunity targeting both T and B cell responses.

immunology↗

bioRxiv2024

Self-reactive B cells traverse a perfect storm of somatic mutagenesis to cause a virus-induced autoimmune disease

The unexplained association between infection and autoimmune disease is strongest for hepatitis C virus-induced cryoglobulinemic vasculitis (HCV-CV). We traced the evolution of the pathogenic rheumatoid factor (RhF) autoantibodies in four HCV-CV patients by deep single cell multi-omic analysis, revealing three sources of B cell somatic mutation converged to drive accumulation of a large disease causing clone. A sensitive method for quantifying low affinity binding revealed three recurring heavy/light chain combinations created by V(D)J recombination bound self IgG but not viral E2 antigen. Whole genome sequencing revealed accumulation of thousands of somatic mutations, at levels comparable to CLL and normal memory B cells, but with 1-2 corresponding to driver mutations found recurrently in B cell leukemia/lymphoma. V(D)J hypermutation created autoantibodies with compromised solubility. In this virus-induced autoimmune disease, infection promotes a perfect storm of somatic mutagenesis in the descendants of a single B cell.

immunology↗

bioRxiv2022

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

immunology↗

bioRxiv2021

Longitudinal characterisation of phagocytic and neutralisation functions of anti-Spike antibodies in plasma of patients after SARS-CoV-2 infection.

Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions. Author SummaryLimited data suggest antibody dependent effector functions including phagocytosis may contribute to the immunological and inflammatory responses in SARS CoV-2 infection, however, their development, maintenance, and clinical significance remain unknown. In this study we show: O_LIPatients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma. C_LIO_LIIn convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1. C_LIO_LILongitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice. C_LI

immunology↗

bioRxiv2021

Ageing impairs the airway epithelium defence response to SARS-CoV-2

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

immunology↗

bioRxiv2020

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies

Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

immunology↗

bioRxiv2020

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, we performed viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. Despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.

genomics↗

bioRxiv2020

Optimised cell systems for the investigation ofhepatitis C virus E1E2 glycoproteins

Great strides have been made in understanding and treating Hepatitis C virus (HCV) thanks in part to the development of the full-length cell-culture system, the pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterising neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones fail to produce infectious particles or produce particles that exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in HEK 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T and 293F exhibit important differences. We found that 293F-produced sE2 harbours mostly complex type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex type glycans and highmannose or hybrid type glycans. Moreover, sE2 produced in HEK 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

microbiology↗

bioRxiv2019

Single cell multi-modal analysis with VDJPuzzle identifies clonality and gene expression profiles of T and B cells

BackgroundSingle cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information.\n\nResultsWe developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians.\n\nConclusionsVDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview.

bioinformatics↗